Transcription is the major control point of gene expression and RNA polymerase (RNAP), conserved from bacteria to man, is the central enzyme of transcription. Our long term goal is to understand the mechanism of transcription and its regulation. Determining three-dimensional structures of RNAP and its complexes with DNA, RNA, and regulatory factors, is an essential step. We focus on highly characterized prokaryotic RNAPs. To this end, we bring to bear a combined biochemical and biophysical approach. Here we propose structure/function studies of transcription complexes in different stages of the transcription cycle, aimed towards adding to our understanding of RNAP regulation by the product of transcription itself, RNA. Specifically, we propose to: 1. Use X-ray crystallography to determine high-resolution structures of Thermus RNAP paused transcription complexes, with and without NusA or NusA domains, at the his pause site. Transcriptional pausing plays key roles in the regulation of gene expression by coordinating RNAP with other regulatory events. Transcriptional pausing couples transcription and translation to control the expression of many amino acid biosynthesis operons in a process called attenuation. These regulatory pauses, such as at the his pause site, are stabilized by an RNA hairpin that forms in the just transcribed RNA transcript, likely through an allosteric mechanism. In addition, extrinsic factors, such as the conserved elongation factor NusA, can further stabilize the pause. We've crystallized a paused elongation complex and collected diffraction data to 3.8 E-resolution. Further experiments are proposed to i) improve the resolution limit of these crystals, ii) trap additional relevant conformational states of the paused complex, and iii) crystallize a complex containing NusA or NusA domains. 2. Structurally characterize the 6S RNA/RNAP-holoenzyme complex. The 6S RNA, a key player in the response of the bacterial transcriptional program to nutrient limitation in stationary phase, binds with marked specificity to C70-holoenzyme and inhibits its function. The 6S RNA mimics the DNA in an open promoter complex, and can serve as a transcription template, providing a mechanism for releasing the 6S RNA when nutrients become plentiful. We will: i) Use biochemical and biophysical approaches to map E. coli C70-holoenzyme interactions with 6S RNA, ii) Use X-ray crystallography to determine structures of 6S RNA/RNAP-holoenzyme complexes.